Sequence specific antimicrobials

ABSTRACT

Provided are compositions and methods for selectively reducing the amount of antibiotic resistant and/or virulent bacteria in a mixed bacteria population, or for reducing any other type of unwanted bacteria in a mixed bacteria population. The compositions and methods involve targeting bacteria that are differentiated from other members of the population by at least one unique clustered regularly interspaced short palindromic repeats (CRISPR) targeted DNA sequence. The compositions and methods can be readily adapted to target any bacteria or any bacteria plasmid, or both.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/766,675, filed on Aug. 7, 2015, which is a National Phase ofInternational Application No. PCT/US2014/015252, filed on Feb. 7, 2014,which claims priority to U.S. Provisional Application No. 61/761,971,filed on Feb. 7, 2013, the disclosures of each of which are incorporatedherein by reference.

FIELD OF THE DISCLOSURE

The present disclosure is related to compositions and methods forselectively killing specific bacteria in mixed bacteria populations.

BACKGROUND OF THE DISCLOSURE

Advances in DNA sequencing technologies have revealed the diversity ofcomplex microbial populations in many different environments. They havealso provided evidence for the contributions that individual speciesmake to the whole of the population and/or to their environment. Perhapsthe most striking example of this is the human microbiome and itsinfluence on human health. The study of the microbiome not only showsthe importance of certain species for the human host, but has alsorevealed the undesired side-effects of traditional antimicrobialswithout killing specificity, which include promoting the emergence ofantibiotic resistance and important negative effects on human health.These examples highlight the ongoing need for tools to accuratelycontrol and manipulate complex microbial consortia. The presentdisclosure meets these and other needs.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions and methods for selectivelyreducing the amount of antibiotic resistant and/or virulent bacteria ina mixed bacteria population, or for reducing any other type of unwantedbacteria in a mixed bacteria population. The compositions and methodsinvolve targeting bacteria, wherein the targeted bacteria can bedifferentiated from other members of the population by at least oneunique clustered regularly interspaced short palindromic repeats(CRISPR) targeted DNA sequence. Various embodiments of the compositionsand methods of the disclosure are demonstrated using Staphylococcusaureus and CRISPR systems that employ nucleotide sequences directed toS. aureus, but the compositions and methods can be readily adapted,given the benefit of the present disclosure, to target any bacteria.

In one aspect the disclosure provides pharmaceutical compositions forselectively reducing the amount of bacteria in a mixed bacteriapopulation wherein the composition comprises a pharmaceuticallyacceptable carrier and a packaged, recombinant phagemid. The phagemidcomprises a CRISPR system, wherein the CRISPR system comprisesnucleotide sequences encoding i) a CRISPR-associated (Cas) enzyme; andii) a targeting RNA selected from at a) least one bacterial chromosometargeting RNA; or b) at least one plasmid targeting RNA; or acombination of a) and b). In embodiments, the Cas enzyme encoded by theCRISPR system is a wild type Cas enzyme, or a modified Cas enzyme. Inembodiments, the targeting RNA is selected from a CRISPR RNA (crRNA) anda guide RNA. If the targeting RNA is a crRNA, the phagemid furthercomprises a sequence encoding a separately transcribed trans-activatingCRISPR crRNA (tracrRNA) sequence. In embodiments, the targeting RNA isdirected to a bacterial virulence gene or an antibiotic resistance genein the bacteria. Such gene targets can be on the bacterial chromosome, aplasmid in the bacteria, or both. In embodiments, the targeting RNA isspecific for a DNA sequence present in a virulent and/or antibioticresistant bacteria, but the DNA sequence is not present in non-virulentand non-antibiotic resistant bacteria in the bacterial population.

In another aspect the disclosure provides methods for reducing theamount of virulent and/or antibiotic resistant bacteria or plasmids in abacterial population. The method comprises contacting the bacterialpopulation with a pharmaceutical composition as described herein,wherein contacting the bacterial population is such that at least someof the phagemids are introduced into at least some of the bacteria inthe bacterial population and, subsequent to the introduction of thephagemids, at least the targeting RNA and the Cas enzyme are produced.Subsequent to production of at least the targeting RNA and the Casenzyme, the amount of unwanted bacteria in the population, such as theamount of virulent and/or antibiotic resistant bacteria, or plasmids inthe mixed bacterial population, is reduced.

In another aspect, the disclosure includes a method for personalizedtherapy for an individual in need of therapy for a bacterial infection.The method comprises obtaining a biological sample from the individualand determining a plurality of bacterial DNA sequences from the sample.The biological sample can be any biological sample, including but notnecessarily limited to a liquid biological sample, such as blood,mucous, serum, cerebrospinal fluid, saliva, and urine, or a solidbiological sample, such as a biopsy of any tissue, or it can comprise asample obtained by using an implement such as a swab. The biologicalsample can be tested directly or it can be processed before determiningthe sequences. Based on determining the sequences one or more virulentand/or antibiotic resistant bacterial species are identified, thusproviding one or more DNA sequences that are unique to those bacteria.Based on the unique DNA sequences a phagemid CRISPR system is developedthat comprises targeting RNA that target the unique sequence(s) presentin the virulent and/or antibiotic resistant bacterial species. Thephagemids are encapsidated (i.e., packaged) in phage proteins and thepackaged phagemids are mixed with a pharmaceutically acceptable carrierto provide a pharmaceutical composition. The pharmaceutical compositionis administered to the individual from which the biological sample wasobtained. The administration is such that at least some of the phagemidsare introduced into at least some of the virulent and/or antibioticresistant bacteria in or on the individual and, subsequent to theintroduction of the phagemids, at least the targeting RNA and the Cas9enzyme are produced, and wherein the amount of virulent and/orantibiotic resistant bacteria and/or the amount of targeted plasmids inthe bacteria is reduced. Thus the method facilitates reducing the amountof pathogenic bacteria on or in the individual, but the amount ofnon-pathogenic bacteria is not reduced.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence-specific killing of S. aureus by aphagemid-delivered CRISPR system. (A) The ΦNM1 phage delivers the pDB121phagemid to S. aureus cells. pDB121 carries the S. pyogenes tracrRNA,cas9 and a programmable CRISPR array sequences. Expression of cas9 and aself-targeting crRNA leads to chromosome cleavage and cell death. (B)Lysates of pDB121 phagemid targeting the aph-3 kanamycin resistance geneor a non-targeting control are spotted on top-agar lawns of eitherRN^(Φ) or RNK^(Φ) cells. (C) Treatment of RN^(Φ) (blue triangles) orRNK^(Φ) (red diamonds) with pDB121::aph at various MOI. Survival iscomputed as the ratio of CFU recovered after treatment over CFU from anuntreated sample of the same culture (mean±s.d.). The black curverepresents the probability that a cell does not receive any phagemidmaking the assumption that all cells have the same chance to receive aphagemid. (D) Time course of the treatment by pDB121::aph ofRNK^(Φ)/pCN57 (GFP reporter plasmid) cells either in a monoculture or ina mixed culture with non-targeted RN^(Φ) cells.

FIG. 2 shows an example of targeting of antibiotic resistance genes andplasmids in a MRSA strain. (A) Treatment of a mixed population resultsin killing of the targeted USA300 MRSA strain and delivery of animmunizing phagemid in the rest of the population. (B) pDB121::mecAspecifically kills USA300^(Φ) in a mixed population. Exponentiallygrowing USA300^(Φ) and RN^(Φ) cells were mixed 1:1 and treated withpDB121 at an MOI of ˜5. Cells were plated either on a non-selectivemedium, on chloramphenicol-containing medium to measure the proportionof cells receiving the phagemid treatment, or on oxacilin-containingmedium to measure the proportion of USA300^(Φ) cells in the population(mean±s.d.). (C) The CRISPR array can be programmed to target the pUSA01and pUSA02 plasmids simultaneously. (D) USA300^(Φ) was treated withpDB121 lysates targeting each plasmid individually or in combination.Cells were plated either on a non-selective medium, onchloramphenicol-containing medium to measure the proportion of cellsreceiving the phagemid treatment, or on tetracycline-containing mediumto measure the proportion of cells being cured from pUSA02 (mean±s.d.).(E) Plasmid curing is confirmed by the lack of PCR amplification withplasmid specific oligonucleotides in 8 independent clones treated withthe double targeting construct. (F) A population of RN^(Φ) cells wasimmunized against plasmid horizontal transfer by treatment with thepUSA02-targeting pDB121 phagemid. 30 min after treatment, the populationis transduced with a ΦNM1 stock grown on USA300. Cells are plated eitherwithout selection or on tetracycline to measure transduction efficiencyof the pUSA02 plasmid (mean±s.d.).

FIG. 3 shows an example of sequence specific killing of kanamycinresistant S. aureus in a mouse skin colonization model. Mice skin wascolonized with a 1:1 mixture of 10⁵ RN^(Φ) and RNK^(Φ) cells carryingthe pCN57 GFP reporter plasmid, followed by treatment at 1 hr witheither pDB121::aph or a non-targeting control phagemid. After 24 hr theskin from the treated area was excised, homogenized and the cells platedon mannitol salt agar (A) Pictures of two representative plates in theGFP channel. (B) The proportion of RNK^(Φ) cells in the population wasmeasured as the proportion of green fluorescent colonies on the plates(mean±s.d.). The p-value of a two tailed student test not assuming equalvariance is reported (n=5).

FIG. 4 shows an example of how the S. pyogenes CRISPR-Cas system can beprogrammed to kill S. aureus. Plasmid pDB114 carries the S. pyogenestracrRNA, cas9 and a minimal array containing two repeats separated by asequence containing BsaI restriction sites used to clone crRNA guidesequences using annealed oligonucleotides. pDB114 was programmed totarget the aph-3 kanamycin resistance gene and transformed either inelectrocompetent RN4220 cells or RNK cells carrying aph-3 in thechromosome. Chloramphenicol resistant CFU obtained in three independentassays are reported (mean±s.d.).

FIG. 5 shows an example of phagemid transduction efficiency. ΦNM1lysates were prepared on RN4220 cells containing either pC194, pDB91 orpDB121. PFU/μl and TU/μl were measured as described in materials andmethods (mean±s.d.).

FIG. 6 shows and example of an analysis of survivor colonies. Eightcolonies of RNK^(Φ) that survived a treatment with pDB121::aph werere-streaked on TSA-chloramphenicol plates. Two chloramphenicol-resistantcolonies were isolated, indicating that they carried the pDB121 plasmid(E3 and E7). (A) The plasmids from E3 and E7 were purified andretransformed in RN4220 or RNK electrocompetent cells and plated onTSA-chloramphenicol. While a control pDB121::aph plasmid did not yieldany colonies when transformed in RNK, plasmids prepared from E3 and E7could be efficiently transformed, indicating that the CRISPR system wasno longer functional. (B) In order to assess the integrity of thetarget, the survivor colonies were streaked on a HIA containing 5 mMCaCl₂ plate and a drop of pDB121::aph phagemid was added on top.Clearance indicates that the isolated colonies are still sensitive;hence the target is still intact.

FIG. 7 shows an example of killing with multiple crRNA guides. Lysatesof the pDB121 phagemid targeting carrying mecA, mecA2 or sek eitheralone or in combination were used to infect USA300 or RNK^(Φ) cells witha MOI of ˜10. Cells were plated on non-selective TSA plates.

FIG. 8 demonstrates shows transcriptional repression using a modifiedCas enzyme. Panel A) provides an illustrative representation of agfpmut2 reporter gene showing positions that were targeted by the Cas9D10A-H840 double mutant for repression of GFP expression. Panel B) showsrepression at positions targeting the top-strand, while Panel C) showstargeting of the bottom-strand.

DETAILED DESCRIPTION OF THE DISCLOSURE

Traditional antimicrobials target conserved cellular pathways andtherefore cannot selectively kill specific members of a complexmicrobial population. However, there are many instances when thesurvival of specific members of the population is desirable, for examplefor the manipulation of the human microbiome. Therefore the ability toremove specific members of a microbial consortium would be desirable,and is achieved by the present disclosure. In particular, we demonstrateseveral representative, programmable, sequence-specific antimicrobialsusing the RNA-guided Cas9 nuclease and a phagemid system for itsdelivery into Staphylococcus aureus.

As is recognized in the art, a CRISPR site is a distinctive DNA locus(i.e., an array or cluster of DNA sequences) found in the genomes ofmany bacteria and archaea. It has been reported that CRISPR sequencescan function as a type of “immune system” that help bacteria defendagainst phage infections (see, for example, Barrangou et al., “CRISPRProvides Acquired Resistance Against Viruses in Prokaryotes,” Science315:1709-12 (March 2007); Deveau et al., J. Bacteriol. 190(4): 1390-1400(February 2008); Horvath et al., J. Bacteriol. 190(4):1401-12 (February2008)).

The present disclosure in part exploits the evolutionary battle betweenphage and bacteria by co-opting the phage to selectively kill specificbacteria in a sequence specific manner that is controlled by deliberateprogramming of the novel phagemid-CRISPR systems described herein. Inthis regard, we show that after programming Cas9 to target virulencegenes, phagemid treatment specifically kills virulent, but notavirulent, staphylococci. Phagemids can also specifically destroystaphylococcal plasmids that spread antibiotic resistance genes andimmunize avirulent cells against the uptake of such plasmids.Sequence-specific killing was also achieved using a murine skincolonization model, demonstrating the broad applicability of Cas9-based,programmable antimicrobials. Additionally, we demonstrate sequencespecific repression of bacterial transcription using a modified Cas9CRISPR system. Thus, the present disclosure provides a widely applicableand adaptable system for selectively killing only certain bacteria in apopulation of bacteria, as well as for eliminating specific geneticelements, such as plasmids, from a population of bacteria, and maintaineradication and/or low levels of such plasmids. Accordingly, given thebenefit of the present disclosure, the compositions and methodsdescribed herein can be readily adapted to selectively kill any bacteriaand/or reduce or eliminate any bacterial extra-chromosomal geneticelements in a mixed population of bacteria to provide a therapeuticand/or prophylactic benefit, or for any other setting or purpose where areduction in specific bacterial populations and/or genetic elementswould be desirable. These include but are not necessarily limited tousing the compositions on medical devices or other medical implements toreduce or eliminate harmful bacteria, or in non-medical applicationswhere it is desirable to inhibit or eliminate specific bacterial growthon non-medical objects or surfaces. Therefore, in general, and as willbe described more fully below, embodiments of the present disclosureprovide pharmaceutical compositions for selectively reducing the amountof antibiotic resistant and/or virulent bacteria in a bacteriapopulation, or for reducing any other type of bacteria in a bacteriapopulation, wherein the targeted bacteria can be differentiated fromother members of the population by way of at least one unique CRISPR DNAsequence. As used herein, bacteria that are virulent and/or antibioticresistant can be considered to be “pathogenic” bacteria.

In embodiments, the composition comprises a packaged, recombinantphagemid, wherein the phagemid comprises a CRISPR system, wherein theCRISPR system comprises nucleotide sequences encoding a Cas enzyme and atargeting RNA selected from at least one chromosome targeting RNA, or atleast one plasmid targeting RNA, or a combination thereof. As describedfurther below, in embodiments, the CRISPR system can be configured totarget only a specific type of bacteria by targeting a specific sequencein the bacteria, such as a DNA sequence present in a virulent and/orantibiotic resistant bacteria, wherein that DNA sequence is not presentin non-virulent and non-antibiotic resistant bacteria that are in thesame bacteria population as the targeted bacteria.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure pertains.

Unless specified to the contrary, it is intended that every maximumnumerical limitation given throughout this description includes everylower numerical limitation, as if such lower numerical limitations wereexpressly written herein. Every minimum numerical limitation giventhroughout this specification will include every higher numericallimitation, as if such higher numerical limitations were expresslywritten herein. Every numerical range given throughout thisspecification will include every narrower numerical range that fallswithin such broader numerical range, as if such narrower numericalranges were all expressly written herein.

The term “bacteria” as used herein refers to any of the prokaryoticmicroorganisms that exist as a single cell or in a cluster or aggregateof single cells.

The term “pharmaceutically acceptable carrier” as used herein refers toa substantially non-toxic carrier for administration of pharmaceuticalsin which the compound will remain stable and bioavailable. Adding apharmaceutically acceptable carrier to other components of thecompositions described herein is considered to yield “pharmaceuticalcompositions.” The pharmaceutically acceptable carrier contained in thepharmaceutical composition can be any carrier which is used inpharmaceutical formulations, examples of which include but are notlimited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch,rubber arable, potassium phosphate, arginate, gelatin, potassiumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,water, syrups, methylcellulose, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. The pharmaceuticalcomposition may further include a lubricant, a humectant, an emulsifier,a suspending agent, and a preservative. Details of suitablepharmaceutically acceptable carriers and formulations can be found inRemington's Pharmaceutical Sciences (19th ed., 1995), which isincorporated herein by reference. The pharmaceutical composition canfurther comprise an additional active ingredient(s), such as anantibiotic.

The pharmaceutical compositions can be provided in the form of pills,tablets, coated tablets, lozenges, capsules, solutions, syrups,emulsions, suspensions, as aerosol mixtures, gels, foams, sols,slurries, ointments, creams or tinctures, and can also include othercomponents, such as liposomes, microsomes, nanoparticles, and any othersuitable vehicle for delivering a packaged phagemid of the disclosure toa subject or to any object or non-living surface wherein inhibition orelimination of specific bacteria is desired.

For administering to a subject in need thereof, the pharmaceuticalcomposition can be administered orally, parenterally, topically,nasally, vaginally, or rectally. Parenterally administration includesintravenous, intra-abdominal, intramuscular, intraperitoneal ortransdermal administration. A suitable dosage amount of thepharmaceutical composition of the present disclosure can vary dependingon pharmaceutical formulation methods, administration methods, thepatient's age, body weight, sex, type and location of bacterialinfection, diet, administration time, administration route, an excretionrate. In embodiments, a pharmaceutical composition can be administeredwith a dosage of 10¹-10¹⁴ PFU/kg (body weight). Such a concentration canbe use, for example, as a single or daily dose for a period of time suchthat the unwanted bacteria are reduced to a satisfactory level or areeliminated.

The term “treating” as used herein includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition, substantially preventing the appearance of clinical oraesthetical symptoms of a condition, or protecting from harmful orannoying stimuli.

The terms “inhibiting” “inhibit” and “inhibition” as used herein areused to refer to reducing the amount or rate of a process, to stoppingthe process entirely, or to decreasing, limiting, or blocking the actionor function thereof. Inhibition can include a reduction or decrease ofthe amount, rate, action function, or process by at least 5%, up to andinclude at least 99% when compared to a suitable reference.

The term “variant” and its various grammatical forms as used hereinrefers to a nucleotide sequence or an amino acid sequence withsubstantial identity to a reference nucleotide sequence or referenceamino acid sequence, respectively. The differences in the sequences maybe the result of changes, either naturally or by design, in sequence orstructure. Natural changes may arise during the course of normalreplication or duplication in nature of a particular nucleic acidsequence. Designed changes may be specifically designed and introducedinto the sequence for specific purposes. Such specific changes may bemade in vitro using a variety of mutagenesis techniques. Such sequencevariants generated specifically may be referred to as “mutants” or“derivatives” of the original sequence. For example, in embodiments, theCas used in the present disclosure is a wild type or mutant Casexpressed recombinantly. The Cas sequence can comprise the wild typeamino acid sequence expressed by any bacteria that encodes a Cas. In oneembodiment, the Cas is a Cas9 having a Cas9 amino acid sequence encodedby Streptococcus pyogenes. In one embodiment, the Cas9 is a variant Cas9that comprises one or more mutations. In an embodiment, the Cas9comprises one or more mutations that lessen or eliminate its nucleaseactivity, but its DNA binding ability is retained. In one embodiment,the mutations comprise a D10A and/or an H840A change in theStreptococcus pyogenes Cas9 amino acid sequence. The reference sequenceof S. pyogenes is available under GenBank accession no. NC 002737, withthe cas9 gene at position 854757-858863. The S. pyogenes Cas9 amino acidsequence is available under number is NP 269215. These sequences areincorporated herein by reference as they were provided on the prioritydate of this application or patent.

In an embodiment, a Cas9 comprising the double mutant D10A and an H840Ais used for sequence specific transcription repression in bacteria isconnection with a CRISPR system as further described herein. Thus, thepresent disclosure provides in various embodiments modified CRISPRsystems.

The present disclosure provides modified CRISPR systems that differ fromthose which naturally occur in bacteria. Previous attempts to providemodified CRISPR systems have been described, such as in WO2013176772.However, in contrast to the present case, these disclosures do notinclude any description of compositions and methods for selectivelyreducing pathogenic, i.e., virulent, and/or antibiotic resistant membersof a bacterial population, and do not include a description of phagemidsor using phagemids to achieve this result.

With respect to CRISPR systems, as will be recognized by those skilledin the art, the structure of a naturally occurring CRISPR locus includesa number of short repeating sequences generally referred to as“repeats.” The repeats occur in clusters and up to 249 repeats have beenidentified in a single CRISPR locus and are usually regularly spaced byunique intervening sequences referred to as “spacers.” Typically, CRISPRrepeats vary from about 24 to 47 bp in length and are partiallypalindromic. The repeats are generally arranged in clusters (up to about20 or more per genome) of repeated units. The spacers are locatedbetween two repeats and typically each spacer has a unique sequencesthat are from about 20-72 bp in length. Many spacers are identical to orhave high homology with known phage sequences. In addition to repeatsand spacers, a CRISPR locus also includes a leader sequence and often aset of two to six associated Cas genes. The leader sequence typically isan AT-rich sequence of up to 550 bp directly adjoining the 5′ end of thefirst repeat. New repeat-spacer units are believed to be almost alwaysadded to the CRISPR locus between the leader and the first repeat.

Generally, it is thought that the proteins encoded by the associated Casgenes act as a bacterial “immune system” that confer resistance againstphages (also referred to as ‘CRISPR interference’). At the molecularlevel, CRISPR interference can be divided into three phases. In theadaptation phase, new repeat-spacer units are incorporated to re-programCRISPR interference against new invasive nucleic acids, allowing thecell to adapt rapidly to the invaders present in the environment. Asdescribed more fully below, during the crRNA biogenesis phase,repeat/spacer arrays are transcribed as a long precursor that is cleavedwithin repeat sequences and processed into small CRISPR RNAs (crRNAs) byCas endoribonucleases. crRNAs retain spacer sequences that specify thetargets of CRISPR interference. In the targeting phase crRNAs are usedas antisense guides in Cas/crRNA ribonucleoprotein complexes that cleavethe nucleic acids of mobile genetic elements carrying a cognatesequence, known as the protospacer, also referred to herein as“spacers.” In addition to spacer-target complementarity, the presence ofa conserved short tri- or tetra-nucleotide sequence, known as theprotospacer adjacent motif (PAM), is involved in CRISPR interference.

As described above, the present disclosure differs in multiple respectsfrom CRISPR systems that exist naturally. In particular, the presentdisclosure provides modified CRISPR systems that are encoded byphagemids. Phagemids are plasmids modified to carry a phage packagingsite and may also encode phage proteins. Phagemids may comprise, ingeneral at least a phage packaging site and an origin of replication(ori). In embodiments, phagemids of the present disclosure encode phagepackaging sites and/or proteins involved in phage packaging. Inembodiments, the phagemids include a packaging site and are packaged bya phage that is intended to be introduced into a population of bacteriaaccording to the methods of this disclosure. In embodiments, the phageis of a type that selectively infects a pathogenic type of bacteria, ora type of bacteria that can have pathogenic and non-pathogenic membersin a mixed bacteria population, or can infect different types ofbacteria in a mixed bacteria population. In embodiments, the phage isspecific for a particular bacterial genus, species or strain. In anembodiment, the phage is specific for a bacterial strain that is amember of one of: Streptococcus, Staphylococcus, Clostridium, Bacillus,Salmonella, Helicobacter pylori, Neisseria gonorrhoeae, Neisseriameningitidis, or Escherichia coli. In embodiments, the phage is specificfor Staphylococcus aureus. In embodiments, the phage is a staphylococcalΦNM1 phage.

In embodiments, the phagemids encode one or more bacteriophage proteins.In one embodiment, the phagemids comprise a combination of the rinA,terS and terL genes. The phagemids provided by the instant disclosureundergo replication, expression of encoded proteins, and packaging. Thepackaging occurs at least in part because of the packaging signals theyencode and by expression of the packaging proteins, either by thephagemids alone, or in conjunction with a suitable helper phage. Whenencapsidated by phage protein the phagemids are considered to be“packaged phagemids” or a “packaged phagemid.” Thus, in embodiments, thepresent disclosure includes isolated phagemids, as well as isolatedpackaged phagemids, and packaged phagemids which are provided as acomponent of compositions, including but not necessarily limited topharmaceutical composition(s). The present disclosure includes allmethods of making phagemids and packaged phagemids that are describedherein.

The recombinant phagemids encode in various embodiments a CRISPR systemthat is engineered to selectively kill only a subset of bacteria orreduce only a specific plasmid within a bacterial population, or to doboth. The system includes a sequence encoding a CRISPR-associated (Cas)enzyme, such as the S. pyogenes Cas9 and/or a modified version of itdescribed above, and a targeting RNA. A “targeting RNA” is an RNA that,when transcribed from the portion of the CRISPR system encoding it,comprises at least a segment of RNA sequence that is identical to (withthe exception of replacing T for U in the case of RNA) or complementaryto (and thus “targets”) a DNA sequence in the bacterial chromosome, or asequence on a plasmid within the targeted bacteria. The CRISPR systemsof the present disclosure can encode more than one targeting RNA, andthe targeting RNAs can be directed to one or more sequences in thebacterial chromosome, or plasmid, or combinations thereof. The sequenceof the targeting RNA thus dictates what is targeted by the CRISPR systemcarried by the phagemids. Accordingly, any distinct CRISPR sequence thatis specific for any particular type of bacteria that would be desirableto kill can be readily designed based on the present disclosure and theknowledge of those skilled in the art. For example, in any bacterialpopulation which comprises or would be suspected to comprise bacteriathat would be desirable to kill, a CRISPR sequence encoding a targetingRNA directed to any CRISPR site unique to those bacteria can bedesigned. Accordingly, a targeting RNA sequence determines the DNAsequence in a bacteria that will be subject to nuclease cleavage by theCas that is part of the CRISPR system(s) described herein.

In embodiments, the disclosure includes CRISPR systems on phagemidswhich target virulent bacteria within a bacteria population. Thebacterial population can comprise one type of bacteria, but withvirulent and non-virulent members, or the bacterial population cancomprise a plurality of bacterial species, with only certain specieshaving virulent and non-virulent members in the population. Inembodiments, a mixed bacteria population comprises at least twodifferent strains or species of bacteria. In embodiments, the mixedbacteria population comprises from between two distinct types ofbacteria, to up to a thousand distinct types of bacteria, or more. Inembodiments, the bacteria population comprises a plurality of bacteriatypes that have been identified as part of the human microbiome, such asthose bacteria that have been identified by the Human Microbiome Project(HMP) Consortium which employed DNA-sequencing to identify and cataloguethe thousands of microorganisms, including bacteria, that make up thehuman microbiota. In embodiments, the population of bacteria are onlypresent in a specific site of an individual, such as the skin, or aparticular location on the skin, or a particular mucosal tissue.

The term “virulent” as used herein means a bacteria that can cause abacterial disease or infection. In embodiments, virulent bacteria arethose that cause a bacterial disease or infection in a human subject whodoes not have a compromised immune system.

Typically, virulent bacteria will produce certain proteins which arereferred to as “virulence factors.” Virulent bacteria aredistinguishable from those bacteria that normally colonize one or moreof a healthy host's tissue and for which they are thus undesirable tokill under ordinary therapeutic circumstances because the lattergenerally do not express virulence factors, or express lower amounts ofvirulence factors relative to virulent bacteria. As discussed above, thepresent disclosure includes in embodiments CRISPR systems which comprisesequences encoding targeting RNA directed to bacterial DNA sequenceswhich encode virulence factors. Such virulence factors include but arenot necessarily limited to bacteria proteins that are involved inpathogenic adhesion, colonization, invasion, or immune responseinhibitors, or toxins. In embodiments, the virulence factors areselected from proteases, lipases, endonucleases, hemolysins, endotoxinsand exotoxins. The sequences of bacterial genes from a wide array ofbacteria types that encode these and other virulence factors are knownin the art. Virulence factors can be encoded on the bacterialchromosome, or on a plasmid in the bacteria, or both. In embodiments,the virulence factor is encoded by a bacterial superantigen gene, suchas a superantigen enterotoxin gene, one non-limiting example of which isthe S. aureus Sek gene. Additional virulence factors for S. aureusinclude but are not limited to cytolitic toxins, such as α-hemolysin,β-hemolysin, γ-hemolysin, leukocidin, Panton-Valentine leukocidin (PVL);exotoxins, such as toxic shock syndrome toxin-1 (TSST-1); enterotoxins,such as SEA, SEB, SECn, SED, SEE, SEG, SEH, and SEI, and exfoliativetoxins, such as ETA, ETB. Homologues of all of these toxins expressed byother types of bacteria are contemplated as virulence gene targets aswell.

In embodiments, a virulent type of bacteria, or in certain cases anon-virulent type bacteria, may also comprise an antibiotic resistantgene. Antibiotic resistance genes carried by a variety of bacteria areknown in the art and the sequences of antibiotic resistance genes in anyparticular bacteria can be determined if desired. In certainnon-limiting embodiments the present disclosure includes CRISPR systemswhich comprise sequences encoding targeting RNA that is directed tobacterial DNA sequences which comprise antibiotic resistance genes. Inembodiments, the resistance gene confers resistance to a narrow-spectrumbeta-lactam antibiotic of the penicillin class of antibiotics. Inembodiments, the resistance gene confers resistance to methicillin(e.g., methicillin or oxacillin), or flucloxacillin, or dicloxacillin,or some or all of these antibiotics. Thus, in one embodiment, the CRISPRsystem is suitable for selectively killing what has colloquially becomeknown as methicillin-resistant S. aureus (MRSA) which in practice refersto strains of S. aureus that are insensitive or have reduced sensitivityto most or all penicillins. In another embodiment, the CRISPR system issuitable for killing vancomycin resistant S. aureus (VRSA). Inembodiments, vancomycin resistant S. aureus may also be resistant to atleast one of linezolid (ZYVOX™), daptomycin (CUBICIN™), andquinupristin/dalfopristin (SYNERCID™).

If in an antibiotic resistance gene is present on a plasmid, and theCRISPR system provided only targets the plasmid (which does notnecessarily mean the antibiotic resistance gene itself is targeted, solong as the plasmid on which it resides is targeted), this disclosurethereby includes converting a population of bacteria from antibioticresistant to an antibiotic sensitive population. If desired anappropriate antibiotic can then be used to rid a subject of all or mostof the bacteria that have accordingly been sensitized to the antibiotic.Additional antibiotic resistant genes include but are not limited tofosfomycin resistance gene fosB, tetracycline resistance gene tetM,kanamycin nucleotidyltransferase aadD, bifunctional aminoglycosidemodifying enzyme genes aacA-aphD, chloramphenicol acetyltransferase cat,mupirocin-resistance gene ileS2, vancomycin resistance genes vanX, vanR,vanH, vraE, vraD, methicillin resistance factor femA, fmtA, mecI;streptomycin adenylyltransferase spc1, spc2, ant1, ant2, pectinomycinadenyltransferase spd, ant9, aadA2.

The targeting RNA encoded by the CRISPR system can be a CRISPR RNA(crRNA) or a guide RNA. The sequence of the targeting RNA is notparticularly limited, other than by the requirement for it to bedirected to (i.e., having a segment that is the same as orcomplementarity to) a CRISPR site that is specific for the type ofbacteria and/or plasmid that is to be killed or eliminated from thebacteria, respectively. In this regard, as described briefly above, atarget sequence in the bacteria comprises a specific sequence on its 3′end referred to as a protospacer adjacent motif or “PAM”. The PAM is inthe targeted DNA, but a targeting RNA directed to a sequence adjacent tothe PAM may or may not have the PAM as a component. In general, thepresent disclosure is pertinent to target spacer sequences that aresubject to cleavage by any Type II CRISPR system, and thus the targetsequences conform to the well-known N12-20NGG motif, wherein the NGG isthe PAM sequence. It will be recognized that 20 nts is the size of thehomology sequence in processed crRNA, but, for example, when using aguide RNA that is not processed, the homology sequence can be more than20 nts, such as up to 40 or more nts. Thus, in embodiments, a targetingRNA used in this disclosure will comprise or consist of a segment thatis from 12-40 nucleotides in length. If the phagemid encodes a crRNA,including but not necessarily limited to a pre-crRNA, the phagemid willalso encode a tracrRNA. In various embodiments, the tracrRNA cancomprise a segment that is complementary to a pre-crRNA, such that aportion of the tracrRNA and pre-crRNA can form an RNA duplex. The RNAduplex is cleaved by RNase III, resulting in the formation of acrRNA/tracrRNA hybrid complex. This hybrid functions as a guide for Cas,which cleaves the target sequence in the bacteria. In general, atracrRNA used in embodiments of the present disclosure will comprise orconsist of from 40 to 200 nucleotides, inclusive, and including allintegers and ranges there between. tracrRNA produced in S. pyogenes is a171 nt RNA which is then processed into 89 or 75 nt fragments. There area wide variety of publicly available resources that can be used todesign suitable tracrRNA sequences and such tracrRNA sequences can beadapted for use with embodiments of the present disclosure. To provideone illustrative embodiment, the S. pyogenes 171 species of the tracrRNAis:

(SEQ ID NO: 30) AGTATTAAGTATTGTTTTATGGCTGATAAATTTCTTTGAATTTCTCCTTGATTATTTGTTATAAAAGTTATAAAATAATCTTGTTGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.The 89 nt species is:

(SEQ ID NO: 31) GTTGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.The 75 nt species is:

(SEQ ID NO: 32) AAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.In an embodiment, the sequence of the mature crRNA comprises 20 nt ofsequence that is homologous to the target, followed by:-GTTTTAGAGCTATGCTGTTTTG (SEQ ID NO:33). To illustrate one non-limitingembodiment, a segment of a CRISPR array comprising two spacers can havethe sequence:

GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC- (spacer 1 sequence (30nt))-GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC- (spacer 2 sequence (30nt))-GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC.  TheGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC  corresponds to SEQ ID NO: 34.To provide one non-limiting example of a repeat-spacer sequence,combining the repeat sequence shown in SEQ ID NO:34 with the aph spacersite as shown in Table 1 as SEQ ID NO:1 yields:

(SEQ ID NO: 39) 5′-TCATGAGTGAGGCCGATGGCGTCCTTTGCT-GTTTTAGAGCTATGCTGTTTTG 3′.RNA forms of these sequences have the T replaced with U.

In general a mature crRNA, meaning a crRNA that is complexed with a Casduring cleavage of a DNA target sequence, will comprise or consist offrom 20-60 nucleotides. In embodiments, a crRNA comprises or consists of20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39 or 40 nt of the spacer (targeting) sequence followed by 19-36 ntof repeat sequence. In specific and non-limiting embodiments, thetargeting RNA will comprise or consist of a segment that targets any oneof the genes for which representative spacer sequences are presented inTable 1. It will be recognized that where T is presented in thesequences of Table 1 it will be replaced by U in the targeting RNA. Thetargeting RNA can therefore comprise a segment that itself comprises orconsists of a sequence that is identical to any of the sequencespresented in Table 1 wherein each T is replace by U.

In embodiments, instead of separately providing for transcription of atracrRNA and a crRNA, the disclosure includes providing a phagemid whichencodes a guide RNA that contains both a tracrRNA segment and a crRNAsegment. This configuration abrogates the requirement for separatetranscription initiation sites, separate promoters, and the like, and asa consequence produces a crRNA-tracrRNA fused hybrid RNA. In general, aguide RNA sequence will comprise or consist of between 40-200nucleotides of tracrRNA sequence. In embodiments, from 40-100nucleotides of tracrRNA is present in a guide RNA. In one embodiment, 85nucleotides of tracrRNA are present in a guide RNA. With respect to thespacer (targeting) segment of a guide RNA or the targeting portion of acrRNA can comprise or consist of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nt of the spacer(targeting) sequence, and may further comprise 19-36 nt of repeatsequence.

In addition to the compositions described above, the present disclosurealso provides methods for selectively reducing the amount of virulentand/or antibiotic resistant bacteria in a bacterial population. Themethod comprises contacting the bacterial population with a compositioncomprising a packaged phagemid, wherein the phagemid encodes a CRISPRsystem as described herein, wherein the contacting the bacterialpopulation is such that at least some of the phagemids are introducedinto at least some of the bacteria in the bacterial population and,subsequent to the introduction of the phagemids, at least the targetingRNA and the Cas enzyme are produced, and wherein the amount of thevirulent and/or antibiotic resistant bacteria in the mixed bacterialpopulation is reduced, while non-virulent and/or non-antibioticresistant bacteria in the population are not reduced from the effects ofthe CRISPR system. In embodiments, the compositions and methods aredirected to selectively reducing virulent and/or antibiotic resistant S.aureus in a mixed population of bacteria. In embodiments, at least50%-99.9% of the targeted bacteria, inclusive, and including allintegers to the first decimal place there between and all ranges therebetween, are killed by practicing an embodiment of this disclosure.

It will be apparent that this disclosure provides broadly applicablecompositions and methods for prophylaxis and/or therapy of diseasesand/or bacterial infections that are caused by or are suspected of beingcaused by known strains of virulent and/or antibiotic resistant bacteriahaving known gene sequences that are suitable for targeting withpre-made CRISPR encoding, packaged phagemid containing compositions.

In another aspect the disclosure includes a method for personalizedprophylaxis and/or therapy of bacterial infections or diseases. Themethod comprises obtaining a sample of a bacterial population from anindividual in need of prophylaxis and/or therapy for a conditionassociated with a bacterial infection, and determining DNA sequences fora plurality of bacterial species in the sample population. By analyzingthe DNA sequences, the presence and/or amount of virulent or otherwiseundesirable bacteria can be determined and a CRISPR system as describedherein can be designed using targeting RNA directed to unique CRISPRsite DNA sequences in the bacteria associated with the condition. TheDNA sequences of the bacteria in the sample can be analyzed using anysuitable technique. As noted above, DNA sequencing has been used toidentify and catalog many bacteria that make up the human microbiota.Further, many sequencing approaches, such as so-called deep sequencing,massively parallel sequencing and next generation sequencing can be usedand such services are offered commercially by a number of vendors. Acomposition comprising a CRISPR system designed to target onlypathogenic bacteria in the sample is administered to the individual suchthat at least some of the phagemids are introduced into at least some ofthe unwanted bacteria in or on the individual and, subsequent to theintroduction of the phagemids, at least the targeting RNA and the Cas9enzyme are produced, subsequent to which the amount of unwanted bacteria(and/or the amount of unwanted plasmids) in or on the individual isreduced. Other, beneficial bacteria are accordingly not affected by theCRISPR system, even if a phagemid is introduced into them.

It will be apparent from the foregoing that various embodiments of thedisclosure provide compositions and methods suitable for treating asubject for any condition that is caused by or is positively correlatedwith the presence of unwanted bacteria. In embodiments, the method issuitable for prophylaxis and/or therapy for a subject for any one orcombination of conditions associated with an infection by any one orcombination of Streptococcus, Staphylococcus, Clostridium, Bacillus,Salmonella, Helicobacter pylori, Neisseria gonorrhoeae, Neisseriameningitidis, or Escherichia coli. In embodiments, the individual is inneed of treatment for or is at risk of contracting a nosocomialinfection. In embodiments, the individual is an immunocompromisedindividual. In embodiments, the individual is in need of treatment foror is at risk for contracting a bacterial infection of the skin and/or amucosal surface. In embodiments, the individual is in need of treatmentfor an infection of S. aureus. In embodiments, the subject is a humanpatient. In embodiments, the compositions and methods are adapted forveterinary medicine purposes for treating non-human animals, includingbut not necessarily limited to canines, felines, equines, and bovines.

In one embodiment, the method for personalized prophylaxis and/ortherapy comprises obtaining a sample from one or more individuals,and/or one or more surfaces, wherein the individuals and/or the surfaceshave been exposed to or are suspected of having been exposed to apathogenic bacteria, identifying CRISPR target sites in the pathogenicbacteria, and administering to the individual a composition comprisingpackaged phagemids which comprise a CRISPR system designed to kill thepathogenic bacteria. In embodiments, the individuals and/or the surfaceswere purposefully exposed to the pathogenic bacteria.

The following examples are presented to illustrate the presentdisclosure. They are not intended to be limiting in any manner. In someaspects, these Examples include routine techniques and methods used inthe field of genetic engineering and molecular biology that are nototherwise described. The following resources include descriptions ofgeneral methodology useful in accordance with the disclosure: Sambrooket al., Molecular Cloning: A Laboratory Manual (4th Ed., 2012);Kreigler, Gene Transfer and Expression: A Laboratory Manual (1993) andAusubel et al., Eds. Current Protocols in Molecular Biology (1995).These general references provide definitions and methods known to thosein the art. However, it is not intended that the present disclosures belimited to any particular methods, protocols, and reagents described, asthese may vary in ways that will be understood by the skilled artisan.

Example 1

In the present disclosure we demonstrate the feasibility of usingpackaged phagemids encoding CRISPR systems as sequence-specificantimicrobials that facilitate the selective killing bacteria within aheterogeneous bacteria population. To demonstrate specific embodimentsof this approach, we used the cas9 gene and its RNA guide/s sequencesusing a phagemid, a plasmid designed to be packaged in phage capsids(FIG. 1A). We demonstrate the general applicability of this approach byusing Staphylococcus aureus. We show that we can selectively killantibiotic-resistant and/or virulent Staphylococcus aureus strains whichfor a variety of well-known reasons are pertinent to public health. Inparticular, staphylococci are at the same time the predominant membersof the human skin microbiota and one of the most common causes ofnosocomial infections. The recent increase in staphylococcalpathogenicity is largely the result of the transfer of antibioticresistance and virulence genes via conjugative plasmids and other mobilegenetic elements that has led to the rise of hospital- andcommunity-acquired methicillin- and vancomycin-resistant Staphylococcusaureus (MRSA and VRSA, respectively) strains that are very difficult totreat. To corroborate that Cas9 cleavage of chromosomal sequences issufficient to kill staphylococci, we inserted Streptococcus pyogenescas9, tracrRNA (trans-activating crRNA, a small RNA required for crRNAbiogenesis) and a minimal CRISPR array optimized for one-step cloning ofcrRNA sequences, into the staphylococcal vector pC194, generatingpDB114. This plasmid was programmed to target the aph-3 kanamycinresistance gene and the resulting construct, pDB114::aph, wastransformed into S. aureus RN4220 and a kanamycin-resistant isogenicderivative, S. aureus RNK. Transformation efficiency of RNK cells was atleast 2 orders of magnitude lower than RN4220 (FIG. 4), demonstratingCas9-mediated killing of staphylococci.

Example 2

In order to develop a phagemid system for Staphylococcus aureus, wecloned a ˜2 kb fragment containing the rinA, terS and terL genes andpackaging site from the staphylococcal ΦNM1 phage on plasmid pC194,obtaining pDB91. To assess the efficiency of packaging of pDB91 in ΦNM1capsids, a transduction assay was performed. RN4220 cells containingpDB91 were infected with ΦNM1, and the lysate was used to transduceRN4220 cells previously lysogenized with ΦNM1, referred to as RN^(Φ).The lysogenic strain is resistant to superinfection which allowsobservation of phagemid transduction while negating the presence ofwild-type phage in the lysate. We determined that a lysate with a titerof 1.6×10⁷ plaque forming units (PFU)/μl contained 3.8×10⁶ transferunits (TU)/μl, suggesting both a high efficiency of phagemid packaging(TU/PFU, 24%) as well as a high TU titer to treat a large bacterialpopulation (FIG. 5). We cloned the CRISPR sequences of pDB114 andpDB144::aph into the pDB91 phagemid to obtain pDB121 and pDB121::aph,respectively. The proportion of phage particles that contained thesephagemids was substantially lower (2.9% for pDB121, FIG. 5) than thatfor pDB91, most likely due to the larger size of pDB121 (10.3 kb vs. 5.3kb), but remains high enough to perform delivery to a large number ofcells. When spotted on a lawn of RNK^(Φ) cells, but not on a RN^(Φ)lawn, pDB121::aph elicited a strong growth inhibition (FIG. 1B).Conversely, the non-targeting pDB121 phagemid did not produce anyinhibition of neither strain. In order to quantify the observed killing,infection experiments were performed at different multiplicity ofinfection (MOI). Here, we define the MOI as the number of TU perrecipient cell. In targeting conditions cell killing is observed whenthe MOI becomes greater than one, while non-targeted cells remainunaffected (FIG. 1C). At an MOI of 20, the survival rate is of 1.1×10⁻⁴.In order to investigate the nature of the survivor cells, coloniesrecovered after treatment were re-streaked on chloramphenicol plates,the resistance marker associated with the pDB121 phagemids. We foundthat 6/8 colonies were still sensitive to chloramphenicol, suggestingthat they either lost the phagemid or did not receive it in the firstplace. A Poisson distribution with a mean of 20 gives a probability of acell receiving no phagemid of only 1.5×10⁻⁹, which is substantiallylower than the survival rate observed. This either suggests that theinjection of the phagemid in the recipient cells is not completelyrandom, i.e. some cells are more likely to receive phagemids thanothers, or that the phagemids can be lost at a low rate. The remaining2/8 chloramphenicol-resistant colonies were further analyzed todetermine whether the aph-3 target and/or the CRISPR system were stillintact. We found that they contained plasmids incapable of CRISPRtargeting (FIG. 6A). None of the cells (8/8) able to escape phagemidtreatment contained target mutations (FIG. 6B), indicating that suchmutations happen at a frequency lower than 1 in 1.3×10⁵ cells.

Example 3

Following treatment with conventional antibiotics that eradicate mostmembers of a bacterial community, the rise and propagation of resistanceis fueled by the lack of competitors in the treated environment. Asshown above, sequence-specific killing is not exempt from the generationof cells that escape treatment, however this strategy has the benefit ofkilling only a subpopulation, leaving other members of the bacterialcommunity and even of the same species to colonize the niche and limitthe propagation of resistant organisms. To demonstrate this effect, weperformed a treatment of RNK^(Φ) in which the pCN57 GFP reporter plasmidwas transformed, either alone or in a co-culture with RN^(Φ). In bothcases, the treatment effectively kills the targeted population, asevidenced by an interrupted increase of OD in the monoculture and GFPsignal in the mixed culture respectively (FIG. 1D). After 7 hours,RNK^(Φ) cells that survived the treatment resume growth in themono-culture whereas the fluorescence signal in the co-culture remainsvery low. This difference is most likely a consequence of the growth ofthe non-targeted RN^(Φ) cells, which out-competes the targeted RNK^(Φ)escaper cells for the medium nutrients, leaving them no opportunity togrow.

Example 4

We sought to use the sequence-specific killing strategy to eradicateMRSA strains from a mixed population of bacteria (FIG. 2A) and thereforewe produced a phagemid targeting the methicillin resistance gene mecA,pDB121::mecA. This phagemid was used to treat the clinical isolate S.aureus USA300^(Φ) in a mixed culture with RN^(Φ) cells (both ΦNM1lysogens). Exponentially growing USA300^(Φ) and RN^(Φ) cells were mixed1:1 and treated with pDB121::mecA at an MOI of ˜5. Cells were platedeither to a non-selective medium, oxacillin-containing medium to measurethe proportion of USA300^(Φ) cells in the population, or onchloramphenicol-containing medium to measure the proportion of cellsreceiving the phagemid treatment (FIG. 2B). The proportion of USA300^(Φ)dropped from 50% before treatment to 0.4% after treatment, while nosignificant drop could be observed in the control experiment using thenon-targeting pDB121 phagemid. An advantage of using Cas9-mediatedkilling is the possibility of programming the nuclease with two or morecrRNA guides in order to target different chromosomal sequences andlimit the rise of resistant clones that escape phagemid treatmentthrough the generation of target mutations. We achieved this byexpanding the CRISPR array carried by the phagemid to produce a secondcrRNA targeting either the superantigen enterotoxin sek gene, or anotherregion of the mecA gene. Both of these phagemids were shown to killUSA300^(Φ) but not RN^(Φ) (FIG. 7), and all survivor colonies isolatedlacked target mutations (not shown). Consistently with the results wedid not observed an additive effect in the killing efficiency of themultiple targeting phagemids, as survivor colonies either do not receivethe phagemid or receive a defective CRISPR system.

Example 5

Plasmids are a predominant source of antibiotic resistance and virulencegenes in pathogenic bacteria. The USA300 strain carries three of suchplasmids, pUSA01-3, with the pUSA02 plasmid conferring tetracyclineresistance to this strain. We designed phagemids that target pUSA01,pUSA02 or both (pUSA03 is unstable, data not shown) and tested them fortheir ability to cure this plasmids from the population (FIG. 2C). Inall cases, treating USA300^(Φ) with the phagemid preparation did notresult in any cell death (FIG. 2D, CFU counts without selection similarto the non-targeting control). However, out of 8 colonies recovered foreach experiment all had lost the targeted plasmid(s) (FIG. 2E). Loss ofpUSA02 could be confirmed by the loss of tetracycline resistance in thetreated cells (FIG. 2D). This demonstrates that delivery of thesequence-specific Cas9 nuclease dramatically reduce the plasmid contentin a bacterial population, without killing the hosts. Many of thesevirulence plasmids are able to transfer horizontally and spreadantibiotic resistance. We showed that pneumococci engineered to harbor achromosomal type II CRISPR system programmed to target antibioticresistance and virulence genes were prevented from acquiring these genesboth in vitro and in vivo in a mouse model of pneumococcal infection. Inthis Example we tested whether phagemid treatment could be used toimmunize naïve staphylococci against pUSA02 transfer. An exponentiallygrowing culture of RN^(Φ) cells was treated with the phagemid targetingthe pUSA02 plasmid or the non-targeting control. After 30 minutes, thecells were infected with a ΦNM1 lysate grown on USA300 cells with theability to transduce pUSA02. Transduction efficiency was measured byselecting for tetracycline resistance. While pUSA02 could readily betransferred to cells treated with the control phagemid, no tetracyclineresistant colonies could be recovered with cells treated with thetargeting phagemid (FIG. 2F), showing an efficient immunization againstplasmid transfer.

Example 6

In order to demonstrate that phagemid treatment can be used toselectively kill staphylococci in vivo, we tested it in a mouse skincolonization model. The backs of CD1 mice were shaved and treated withdepilatory cream to expose the skin. An area on the back was colonizedwith 10⁵ cells of a 1:1 mixture of RN^(Φ) and RNK^(Φ) bacteria, thelatter harboring the pCN57 plasmid to facilitate detection of targetedcells by measuring green fluorescence. Following colonization, infectedareas were treated with pDB121::aph or pDB121 and after 24 hr thetreated skin was dissected and homogenized to enumerate staphylococci.The proportion of RNK^(Φ) cells was measured as the ratio of GFPcolonies in the population (FIG. 3A). After treatment the proportion ofRNK^(Φ) cells dropped from 50% to 4.8% and plating the cells onchloramphenicol showed that the phagemid was delivered to 89% of thecells (FIG. 3B). Unexpectedly, a smaller drop was also observed for micetreated with the control phagemid, possibly due to a fitnessdisadvantage of RNK^(Φ)/pCN57 cells when growing on the mouse skin.Thus, in this and the foregoing Examples, we demonstrate the use ofprogrammable Cas9 nuclease activity as a sequence-specific antimicrobialand as a tool to manipulate heterogeneous bacterial populations. Besidesthe aspect of selective killing, provided with a suitable deliverysystem, the built-in multiplex feature of CRISPR-Cas systems could beexploited to target several different species at the same time and/orseveral sequences of the same bacterium to prevent the rise of resistantmutants. This approach can also be used to cure plasmids and othermobile genetic elements from a population without killing the host.Moreover, based on the present disclosure, the technology is easilyadapted to repress the expression of antibiotic resistance, virulenceand other genes of interest without causing the death of the host usingdCas9, the nuclease-defective version of Cas9, as demonstrated herein inExample 7. These unique features create opportunities for theapplication of this technology in many medical, environmental andindustrial settings.

Example 7

Double stranded cleavage of the target by Cas9 is achieved through theaction of a RuvC domain on one strand and HNH domain on the otherstrand. We demonstrated that D10A and a H840A mutation in theserespective domains abolish cleavage. In this Example we used theD10A-H840 double mutant, referred here as Cas9**, to directtranscriptional repression. Cas9** was direct to bind various positionin the promoter and gene of a GFP reporter. See FIG. 8, where panel (b)shows repression at positions targeting the top-strand, and panel (c)shows targeting of the bottom-strand.

Example 8

The Example provides a description of the materials and methods used toobtain the results described in the foregoing Examples.

Strains and culture conditions. S. aureus strain RN4220 was grown at 37C in TSB, when appropriate, with the following antibiotics: kanamycin(Kan, 25 μg/ml), chloramphenicol (Cm, 10 μg/ml) and tetracycline (Tet, 5μg/ml). S. aureus USA300 was provided by the Fischetti lab. Phage ΦNM1was isolated from the S. aureus Newman strain. The supernatant of anovernight culture was used to infect RN4220 in a top-agar layer. Singleplaques were isolated and passaged 3 times to ensure purity. ΦNM1lysogens of RN4220 (RN^(Φ)) and USA300 (USA^(Φ)) were isolated byre-streaking cells from the middle of a turbid plaque twice. Chromosomalintegration of ΦNM1 was checked by both PCR and by ensuring resistanceto ΦNM1 superinfection. A kanamycin resistance gene was introduced inRN4220 by using a derivative of the pCL55-itet integrative vector wherethe chloramphenicol resistance gene was replaced with the aphA-3kanamycin resistance gene to produce pKL55-itet. Briefly, aphA-3 wasamplified from strain crR6 using primers L484/L485 and pCL55-itet wasamplified with primers L482/L483, followed by digestion with XhoI andligation. Integration in the RN4220 chromosome was achieved bytransformation in electrocompetent cells and selection on TSA+Kan.

Plasmid construction. To assemble the pDB91 phagemid, the rinA-terS-terLregion of ΦNM1 was amplified with oligos B234/B235, and pC194 witholigos B233/B127. PCR products were digested with KpnI and SphI followedby ligation and transformation in RN4220 competent cells. The pDB114plasmid was constructed in two steps. First the full M1GAS S. pyogenesCRISPR02 system was cloned on pC194 by amplifying S. pyogenes genomicDNA with oligos L362/W278 and pC194 with oligos W270/W282, followed bydigestion with BglII and BssSI and ligation, giving pWJ40. The pWJ40plasmid was then amplified with oligos B334/L410 and the BsaI CRISPRarray from pCas9 with oligos L409/B333, followed by Gibson assembly. Toconstruct pDB121, pDB114 was amplified with oligos B351/W278 and pDB91with oligos L316/L318, followed by Gibson assembly of the two fragments.Spacers were cloned by digestion with BsaI, and ligation of annealedoligonucleotides designed as follow: 5′-aaac+(target sequence)+g-3′ and5′-aaaac+(reverse complement of the target sequence)-3′, where thetarget sequence is 30 nt and is followed by a functional PAM (NGG).Alternatively, two spacers were cloned in a single reaction in the BsaIdigested pDB121 vector. Two pairs of oligonucleotides were annealed andligated with the vector. The pair carrying the first spacer was designedas follow:

(SEQ ID NO: 35) 5′-aaac + (target sequence) + GTTTTAGAGCTATG-3′ and (SEQ ID NO: 36) 5′-AACAGCATAGCTCTAAAAC + (reverse complement ofthe target sequence)-3′,  and the pair carrying the second spacer as follow:  (SEQ ID NO: 37)5′-CTGTTTTGAATGGTCCCAAAAC + (target sequence) + g-3′   and(SEQ ID NO: 38) 5′-aaaac + (reverse complement of the target sequence) +GTTTTGGGACCATTCAA-3′.A list of all spacers tested in this study is provided in Table 1, and alist of oligonucleotides in Table 2.

Phage and phagemid production. Phage and phagemid stocks were producedby growing cells from an overnight culture diluted 1:50 in TSB+Cm+CaCl25 mM until an OD600 of 0.6. The cultures were then inoculated with 10 μlof a concentrated ΦNM1 phage stock and incubated for 3H. Cell debris waseliminated by centrifugation and filtering of the supernatant through0.45 um filters. Phage titers were determined by serial dilution andspotting on a top-agar layer of RN4220 cells on HIA plates supplementedwith 5 mM CaCl2. To determine the transducing titer, serial dilutions ofthe phage stock were produced and used to infect a culture of RN^(Φ)cells grown to OD-1. After 1H of incubation at 37 C, cells were platedon TSA+Cm, and transducing units (TU) were measured from the number ofCFU obtained.

Killing and plasmid curing assays. Phage stocks were produced on RN4220cells carrying a phagemid with the desired CRISPR spacer. Recipientcells were grown in TSB to an OD600 of 0.6, diluted 10× in TSB+5 mMCaCl2 and 100 μl of the culture was mixed with 100 μl of the appropriatephage stock dilution. After 1H of incubation, cells were plated on TSA.Survival rates were measured as the ratio of CFUs obtained withtreatment over CFUs obtained without treatment. When appropriate, cellswere also plated on TSA+Cm to measure phagemid transduction efficiency,TSA+Tet to measure pUSA02 curing, and TSA+Oxa to measure the proportionof MRSA cells in the population.

Immunization assay. RN^(Φ) cells were diluted 1:100 in TSB and grown toOD600 of 0.2. Phagemid was added to an MOI of 10, and cells wereincubated 30 min to allow for establishment of the CRISPR system. ThepUSA02 plasmid was transduced by infecting with a phiNM1 stock grown onUSA300 cells. Cells were plated on TSB, TSB+Cm or TSB+Tet to measuretransduction efficiency.

Growth curves and fluorescence measurements. Growth curves and GFPfluorescence were measured in a Tecan microplate reader. Cultures werestarted by diluting a ON culture 1:100 in 200 μl of TSB. Phagemid wasadded to an MOI of ˜10 after 80 min of growth.

Mouse skin colonization. The Rockefeller University's InstitutionalAnimal Care and Use Committee approved all in vivo protocols. Allexperiments were conducted at The Rockefeller University's Animalhousing facility, an AAALAC accredited research facility with allefforts to minimalize suffering. An adapted approach from Kugelberg etal. and Pastagia et al. was used to induce topical skin colonizationwith S. aureus on 6- to 8-week-old female CD1 mice (Charles RiverLaboratories, Wilmington, Mass.). Briefly, mice were anesthetized byintraperitoneal injection of ketamine (1.5 mg/animal; Fort Dodge AnimalHealth, Fort Dodge, Iowa) and xylazine (0.3 mg/animal; Miles Inc.,Shawnee Mission, Kans.). A 2 cm² area of the dorsum of each mouse wasshaved with an electric razor; Nair depilatory cream was then applied tothe shaved area for one minute and wiped away with 70% ethanol pads. Thearea was then tape stripped, with autoclave tape, approximately 10 timesin succession, using a fresh piece of tape each time to irritate andremove the upper layers of the epidermis. The mice were topicallycolonized with a 2 μl mixture of cultures of S. aureus RN^(Φ); andRNK^(Φ)/pCN57 containing 1×10⁵ cells in logarithmic growth phases inPBS. Animals were then immobilized under isoflurane anesthesia. After1H, 10 μl of concentrated phagemid lysate containing 2×10⁷ TU/μl wasapplied on the infected skin area. To obtain this concentration, crudelysates were concentrated using 100 kD Amicon Ultra centrifugal filtersand washed once with PBS. After 24H, tissue from the infected skin areawas excised and homogenized in 0.5 ml of PBS using the Stomacher 80.Bacterial dilutions were plated on mannitol salt agar (an S.aureus-selective medium) and TSA+Cm.

TABLE 1 Spacers used in this disclosure. Target Spacer sequence (5′-3′)aph TCATGAGTGAGGCCGATGGCGTCCTTTGCT  (SEQ ID NO: 1) mecATTTTGAGTTGAACCTGGTGAAGTTGTAATC  (SEQ ID NO: 2) mecA2CATTTTCTTTGCTAGAGTAGCACTCGAATT  (SEQ ID NO: 3) sekGATTATCAATTCCTATATCACCTTGAGCGC  (SEQ ID NO: 4) pUSA01CTTATGTAACTTCAAATAGCCTTCATCAGT  (SEQ ID NO: 5) pUSA02AGGAGTAGTATTAAAATGATTTGCAATATC  (SEQ ID NO: 6)

TABLE 2 Oligonucleotides used in this disclosure. Name Sequence (5′-3′)B234 TTTAGGTACCAAGAGCGAGAGATAGAGATATTAAG  (SEQ ID NO: 7) B235TTTAGCATGCCTATAATCCTAGAGATTTTATTGTGT  (SEQ ID NO: 8) B127AAAAGCATGCAAATATGAGCCAAATAAATATATTC  (SEQ ID NO: 9) B233TACTGGTACCTTTAAAAGCTTCTGTAGGTTTTTAG  (SEQ ID NO: 10) L362aaactcgtgGATTCTGTGATTTGGATCCTTCC  (SEQ ID NO: 11) W278aaaaagatctTATGACTGTTATGTGGTTATCG  (SEQ ID NO: 12) W270aaaaagatctTGCATAATTCACGCTGACCTC  (SEQ ID NO: 13) W282aaaacacgagCGTTTGTTGAACTAATGGGTGC  (SEQ ID NO: 14) L410CTTCACTTGGAACGTTATCCGATTTACCACG  (SEQ ID NO: 15) L409CGTGGTAAATCGGATAACGTTCCAAGTGAAG  (SEQ ID NO: 16) B333CTTTATCCAATTTTCGTTTGAACTCAACAAGTCTCAGTGTGC TG (SEQ ID NO: 17) B334ACACTGAGACTTGTTGAGTTCAAACGAAAATTGGATAAAGTG GG (SEQ ID NO: 18) L316TTAAGGGTTCTTCTCAACGCAC (SEQ ID NO: 19) L318TTAAAAGTTATTGTGATGACGACG (SEQ ID NO: 20) B351ATCGTTTATCGTCGTCATCACAATAACTTTTAAAGATCTTGCATAATTCACGCTGAC (B351 is SEQ ID NO: 21) L482aaaCTCGAGCTGAGAGTGCACCATATGCGG  (SEQ ID NO: 22) L483aaaCTCGAGCTTAATAGCTCACGCTATGCCG  (SEQ ID NO: 23) L484aaaCTCGAGCGCGCAAGCTGGGGATCCG  (SEQ ID NO: 24) L485aaaCTCGAGTAGGTACTAAAACAATTCATCCAG  (SEQ ID NO: 25) B628AGTGGGAAACAACGCCCATGGAG (SEQ ID NO: 26) B629GTTGAACGCATAAATCCAACAAG (SEQ ID NO: 27) B632AGTCACCTCAAGTAAAGAGGTAA (SEQ ID NO: 28) B633TGAAGGACCTAACCCTTCACCTA (SEQ ID NO: 29)

While the disclosure has been particularly shown and described withreference to specific embodiments (some of which are preferredembodiments), it should be understood by those having skill in the artthat various changes in form and detail may be made therein withoutdeparting from the spirit and scope of the present disclosure asdisclosed herein.

What is claimed is:
 1. A pharmaceutical or veterinary composition forselectively reducing the amount of a targeted bacteria in a mixedbacterial population, the composition comprising a pharmaceuticallyacceptable carrier and a packaged, recombinant phagemid, wherein thephagemid comprises a clustered regularly interspaced short palindromicrepeats (CRISPR) system, wherein the CRISPR system comprises nucleotidesequences encoding: i) a CRISPR-associated enzyme; and ii) a targetingRNA selected from: a) at least one bacterial chromosome targeting RNA;b) at least one bacterial plasmid targeting RNA; and c) a combination ofa) and b); wherein the targeted bacteria is an antibiotic resistantbacteria, a virulent bacteria or any unwanted bacteria.
 2. Thepharmaceutical composition of claim 1, wherein the targeting RNA isdirected to a bacterial virulence gene or an antibiotic resistance genein the targeted bacteria.
 3. The pharmaceutical composition of claim 1,wherein the targeting RNA is specific for a DNA sequence present in thetargeted bacteria and wherein said DNA sequence is not present in theother bacteria in the mixed bacterial population.
 4. The pharmaceuticalcomposition of claim 1, wherein the CRISPR-associated enzyme is a Cas9enzyme
 5. The pharmaceutical composition of claim 4, wherein the Cas9enzyme is a Streptococcus pyogenes Cas9.
 6. The pharmaceuticalcomposition of claim 1, wherein the recombinant phagemid is packaged ina phage capsid.
 7. The pharmaceutical composition of claim 2, whereinthe targeted bacteria is any one or a combination of Streptococcus,Staphylococcus, Clostridium, Bacillus, Salmonella, Helicobacter pylori,Neisseria gonorrhoeae, Neisseria meningitidis, or Escherichia coli.
 8. Amethod for selectively reducing the amount of a targeted bacteria in amixed bacterial population, the method comprising: contacting thebacterial population with a pharmaceutical composition comprising apharmaceutically acceptable carrier and a packaged, recombinantphagemid, wherein the phagemid comprises a clustered regularlyinterspaced short palindromic repeats (CRISPR) system, wherein theCRISPR system comprises nucleotide sequences encoding: i) aCRISPR-associated enzyme; and ii) a targeting RNA selected from: a) atleast one bacterial chromosome targeting RNA, b) at least one bacterialplasmid targeting RNA; and c) a combination of a) and b); wherein thetargeted bacteria is an antibiotic resistant bacteria, a virulentbacteria or any unwanted bacteria; wherein the contacting the bacterialpopulation is such that at least some of the phagemids are introducedinto at least some of the bacteria in the bacterial population and,subsequent to the introduction of the phagemids, at least the targetingRNA and the Cas9 enzyme are produced, and wherein the amount of thetargeted bacteria in the mixed bacterial population is reduced.
 9. Themethod of claim 8, wherein the targeting RNA is directed to a bacterialvirulence gene or an antibiotic resistance gene in the targetedbacteria.
 10. The method of claim 8, wherein the targeting RNA isspecific for a DNA sequence present in the targeted bacteria and whereinsaid DNA sequence is not present in the other bacteria in the mixedbacterial population.
 11. The method of claim 8, wherein theCRISPR-associated enzyme is a Cas9 enzyme
 12. The method of claim 11,wherein the Cas9 enzyme is a Streptococcus pyogenes Cas9.
 13. The methodof claim 8, wherein the recombinant phagemid is packaged in a phagecapsid.
 14. The method of claim 8, wherein the targeted bacteria is anyone or a combination of Streptococcus, Staphylococcus, Clostridium,Bacillus, Salmonella, Helicobacter pylori, Neisseria gonorrhoeae,Neisseria meningitidis, or Escherichia coli.